Structure and mechanism of human diacylglycerol O-acyltransferase 1.

Diacylglycerol O-acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans1. DGAT1 belongs to the membrane-bound O-acyltransferase (MBOAT) superfamily, members of which are found in all kingdoms of life and are involved in the acylation of lipids and proteins2,3. How human DGAT1 and other mammalian members of the MBOAT family recognize their substrates and catalyse their reactions is unknown. The absence of three-dimensional structures also hampers rational targeting of DGAT1 for therapeutic purposes. Here we present the cryo-electron microscopy structure of human DGAT1 in complex with an oleoyl-CoA substrate. Each DGAT1 protomer has nine transmembrane helices, eight of which form a conserved structural fold that we name the MBOAT fold. The MBOAT fold in DGAT1 forms a hollow chamber in the membrane that encloses highly conserved catalytic residues. The chamber has separate entrances for each of the two substrates, fatty acyl-CoA and diacylglycerol. DGAT1 can exist as either a homodimer or a homotetramer and the two forms have similar enzymatic activity. The N terminus of DGAT1 interacts with the neighbouring protomer and these interactions are required for enzymatic activity.

Analgesic Buxus alkaloids with Enhanced Selectivity for the Low-Voltage-Gated Calcium Channel Cav 3.2 over Cav 3.1 through a New Binding Mode.

Low-voltage-gated calcium channels (LVGCCs; Cav 3.1-3.3) represent promising drug targets for epilepsy, pain, and essential tremor. At present, modulators with heightened selectivity for a subtype of LVGCCs are still highly desired. In this study we explored three classes of Buxus alkaloids and identified 9(10/19)abeo-artanes Buxusemine H and Buxusemine L (BXSL) as an unprecedented type of Cav 3.2 inhibitors. Particularly, BXSL exhibited Cav 3.2 inhibition comparable to Z944, a non-subtype-selective LVGCCs inhibitor under clinical trial. While lacking specificity for Cav 3.3, BXSL showed a 30-fold selectivity of Cav 3.2 over Cav 3.1. As compared to several well-known inhibitors, the experimental and computational studies suggested BXSL exhibits a distinct binding mode to Cav 3.2, notably through the essential interaction with serine-1543 in domain III. Furthermore, BXSL showed minimal impact on various recombinant and native nociceptive ion channels, while significantly reducing the excitability of isolated mouse dorsal root ganglion neurons. Animal studies in wild-type and Cav 3.2 knock-out mice revealed that BXSL (5 mg/kg), by inhibiting Cav 3.2, exhibits an analgesic effect equivalent to Z944 (10 mg/kg) or mibefradil (10 mg/kg). Moreover, we proposed a structural rationale for the high selectivity of 9(10/19)abeo-artane-type alkaloids towards Cav 3.2 over Cav 3.1. This study introduces a novel analgesic agent and valuable molecular insight for structure-based innovative Cav 3.2 drug development.

Structurally modified Cyclovirobuxine-D Buxus alkaloids as effective analgesic agents through Cav3.2 T-Type calcium channel inhibition.

Cyclovirobuxine-D (CVB-D) is a Buxus alkaloid and a major active constituent in the Chinese medicinal herb Buxus microphylls. Traditionally, the natural alkaloid cyclovirobuxine-D has a long history of use as a traditional Chinese medicine for cardiovascular diseases as well as to treat a wide variety of medical conditions. As we found that CVB-D inhibited T-type calcium channels, we designed and synthesized a variety of fragments and analogues and evaluated them for the first time as new Cav3.2 inhibitors. Compounds 2-7 exhibited potency against Cav 3.2 channels, and two of them were more active than their parent molecules. As a result of the in vivo experiments, both compounds 3 and 4 showed significantly reduced writhes in the acetic acid-induced writhing test. Studies of molecular modeling have identified possible mechanism(s) of Cav3.2 binding. Moreover, the relationship between structure and activity was studied in a preliminary manner. Our results indicated that compounds 3 and 4 could play an important role in the discovery and development of novel analgesics.

Indole-3-acetonitrile Is a Critical Molecule with Weed Allopathic Suppression Function in Broccoli (Brassica oleracea var. italica).

Cruciferous plants are frequently used for ecologically benign weed control in agricultural production. Most effective Broccoli varieties were screened using the entropy method-based topsis model at first. Result showed that varieties of Lvwawa and Lvbaoshiwere most effective in allelopathic suppression on radishes. Column and thin-layer chromatography were used to extract the allelopathic compounds from broccoli residues, which contained various herbicidal active substances; among them, purified single-molecule indole-3-acetonitrile has a stronger inhibitory effect than pendimethalin (commercial herbicide). The weed inhibition rate increased with increasing broccoli residue dosage, with a 40 g/m2 broccoli residue dose yielding the highest suppression rate. Its effect was similar to that of indole-3-acetic acid. Too much of this substance leads to the plant's death. Moreover, broccoli residues had effective control effect on weeds in natural soils in greenhouse and field trials. The results demonstrated that broccoli residue could be used for weed management in field for abundant allopathic suppression molecules to weeds, and that Indole-3-acetonitrile is one of the most important allopathic molecule.

Icetexane diterpenoids as Cav3.2 T-type calcium channel inhibitors from Salvia prattii and analgesic effect of their Semi-synthesized derivatives.

Ten new icetexane diterpenoids, salpratins E-N (1-10) and a known analogue (11) were characterized from Salvia prattii Hemsl. Structurally, 1 is the first 19(4 â†’ 3)-abeo-icetexane diterpenoid featuring with a 6/7/6 ring system. The structures of isolated compounds were determined by comprehensive analyses of spectroscopic data, ECD calculation, and single-crystal X-ray diffraction. Biological studies initially revealed that 1, 7, 10, and 11 are notable Cav3.2 T-type Ca2+ channel (TTCC) inhibitors with IC50 values of 2.9, 5.1, 2.3, and 3.2 µM, respectively. Five icetexane related derivatives (13-17) were synthesized from an abietane type precursor, (+)-carnosic acid (12), for the purpose of overcoming the poor water solubility of aforementioned active compounds and further investigating diverse diterpenes with valuable activity. Among them, 13 and 14 showed potent inhibitions on Cav3.2, having IC50 values of 6.7 and 2.4 µM, respectively. Significantly, they exhibited dose-dependent (1, 3, and 10 mg/kg) and comparable analgesic effects as that of Z944, a TTCCs inhibitor under clinical trial for pain management, in the mouse acetic acid writhing test. These findings further enrich structural diversity and bioactivity of Salvia diterpenoids, as well as provide promising structural templates for the development of Cav3.2 analgesics.

Structure of an EIIC sugar transporter trapped in an inward-facing conformation.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports sugar into bacteria and phosphorylates the sugar for metabolic consumption. The PTS is important for the survival of bacteria and thus a potential target for antibiotics, but its mechanism of sugar uptake and phosphorylation remains unclear. The PTS is composed of multiple proteins, and the membrane-embedded Enzyme IIC (EIIC) component transports sugars across the membrane. Crystal structures of two members of the glucose superfamily of EIICs, bcChbC and bcMalT, were solved in the inward-facing and outward-facing conformations, and the structures suggest that sugar translocation could be achieved by movement of a structured domain that contains the sugar-binding site. However, different conformations have not been captured on the same transporter to allow precise description of the conformational changes. Here we present a crystal structure of bcMalT trapped in an inward-facing conformation by a mercury ion that bridges two strategically placed cysteine residues. The structure allows direct comparison of the outward- and inward-facing conformations and reveals a large rigid-body motion of the sugar-binding domain and other conformational changes that accompany the rigid-body motion. All-atom molecular dynamics simulations show that the inward-facing structure is stable with or without the cross-linking. The conformational changes were further validated by single-molecule Föster resonance energy transfer (smFRET). Combined, these results establish the elevator-type mechanism of transport in the glucose superfamily of EIIC transporters.

The role of splenectomy in lipid metabolism and atherosclerosis (AS).

The extensive performance of splenectomy worldwide for patients suffered from splenic trauma has given rise to high risks of postoperative complications, which has been attracting increasing attention in recent years. Nowadays the spleen is regarded as a versatile organ of the human body, invested with various excellent properties. The spleen has been recognized to take a great part in lipid metabolism. While removal of the spleen intends to alter lipid values, especially with an elevated LDL, splenic autotransplantation is able to normalize these lipid alterations. What is more, conservative surgical procedures like subtotal or partial splenectomy, could as well, afford a correction of dyslipidemia. At the same time, clinically, splenectomy demonstrates a high rate of atherosclerosis (AS), whereas non-surgical treatment after splenic trauma shows unchanged propagation of AS. Based on the intimate relationship between serum lipids and AS, the lipid changes modulated by splenectomy are believed to be responsible for the development of AS. Therefore, a "splenic factor" is most likely present in the regulation of lipidation and AS. Several theories have been postulated to elucidate the possible mechanism involved, among which most are primarily based on its forceful natural immune function, that is to say, the mononuclear phagocytic system.However, the accurate mechanisms behind this mysterious phenomenon still remain unclear so far. Of importance, lipid fractions should be monitored consecutively in case of inevitable splenectomy.

Triterpenes from the aerial parts of Cimicifuga yunnanensis and their antiproliferative effects on p53(N236S) mouse embryonic fibroblasts.

Nine new triterpene derivatives, yunnanterpenes A-F (1-6), 15,16-seco-cimiterpenes A and B (7, 8), and cimilactone C (9), and 15 known analogues (10-24) were isolated from the aerial parts of Cimicifuga yunnanensis. The new structures were established using a combination of MS, NMR, and single-crystal X-ray diffraction techniques. WT MEFs (wild-type mouse embryonic fibroblasts) and tumorigenic cell lines p53(-/-)+H-RasV12 and p53(-/-)+p53(N236S)+H-RasV12 were used for evaluating active structures, targeting p53(N236S) (corresponding to p53(N239S) in humans) mutation. Compound 5 showed nonselective activities against these cell lines, with IC50 values of 5.8, 8.6, and 6.0 µM, respectively. Compound 4 exhibited greater selectivity against the p53(-/-)+p53(N236S)+H-RasV12 cells (IC50 5.5 µM) than against the WT MEFs cells (IC50 14.3 µM).

Cytotoxic cycloartane triterpenes of the traditional Chinese medicine "shengma" (Cimicifuga dahurica).

Twelve new 9,19-cycloartane triterpenes (1-12), together with fourteen known compounds (13-26), were isolated from the roots of Cimicifuga dahurica. Their structures were determined by application of spectroscopic analyses and chemical methods. Biological evaluation of the compounds against human HL-60, SMMC-7721, A549, MCF-7, and SW-480 cell lines indicated that cimigenol-type glycosides (1-3, 19, and 20) showed broad-spectrum and moderate cytotoxicities, with IC50 values ranging from 4.2 to 14.5 µM. Meanwhile, cimigenol-type aglycones (6-8, 15, 16, and 18) exhibited broad-spectrum and week cytotoxicities, having IC50 values around 20 µM. In addition, the key points of the structure-activity relationships of aglycones with a cimigenol skeleton were discussed.

Isolation and Identification of Herbicidal Active Compounds from Brassica oleracea L. and Exploration of the Binding Sites of Brassicanate A Sulfoxide.

Brassica oleracea L. has strong allelopathic effects on weeds. However, the allelochemicals with herbicidal activity in B. oleracea L. are still unknown. In this study, we evaluated the activity of allelochemicals isolated from Brassica oleracea L. based on the germination and growth of model plant Lactuca sativa Linn., grass weed Panicum miliaceum, and broadleaf weed Chenopodium album. Additionally, we employed molecular docking to predict the binding of brassicanate A sulfoxide to herbicide targets. The results of this study showed that eight compounds with herbicidal activity were isolated from B. oleracea L., and the predicted results indicated that brassicanate A sulfoxide was stably bound to dihydroxyacid dehydratase, hydroxymethylpyruvate dioxygenase, acetolactate synthase, PYL family proteins and transport inhibitor response 1. This research provides compound sources and a theoretical foundation for the development of natural herbicides.

The Aqueous Extract of Brassica oleracea L. Exerts Phytotoxicity by Modulating H2O2 and O2- Levels, Antioxidant Enzyme Activity and Phytohormone Levels.

Allelopathic interactions between plants serve as powerful tools for weed control. Despite the increasing understanding of the allelopathic mechanisms between different plant species, the inhibitory effects of B. oleracea on weed growth remain poorly understood. In this study, we conducted experiments to demonstrate that B. oleracea extract can suppress the germination of Panicum miliaceum L.varruderale Kit. seeds as well as of the roots, shoots and hypocotyl elongation of P. miliaceum seedlings. Furthermore, we observed that B. oleracea extract reduced the levels of hydrogen peroxide and superoxide anion in the roots while increasing the activities of catalase and ascorbate peroxidase. In the shoots, B. oleracea extract enhanced the activities of superoxide dismutase and peroxidase. Moreover, the use of the extract led to an increase in the content of phytohormones (indole-3-acetic acid, indole-3-acetaldehyde, methyl indole-3-acetate, N6-isoPentenyladenosine, dihydrozeatin-7-glucoside, abscisic acid and abscisic acid glucose ester) in P. miliaceum seedlings. Interestingly, the aqueous extract contained auxins and their analogs, which inhibited the germination and growth of P. miliaceum. This may contribute to the mechanism of the B. oleracea-extract-induced suppression of P. miliaceum growth.

Studies on the constituents of Cimicifuga foetida collected in Guizhou Province and their cytotoxic activities.

Two new triterpenoids and a chromone glycoside, namely, 24-epi-cimigenol-3-one (1), foetinoside (2), cimifugin-4'-O-[6″-feruloyl]-ß-D-glucopyranoside (3), together with 18 known compounds, were isolated from the rhizomes of Cimicifuga foetida L. collected in Guizhou Province, China. All of the compounds were identified by spectroscopic methods, as well as chemical methods. In the in vitro cytotoxicity evaluation of these compounds against 5 human cancer cell lines, cimigenol (8) exerted the most potent cytotoxic activity against SMMC-7721 (7.87 µM) and A-549 (12.16 µM), while cimiacerin B (9) also showed obvious cytotoxicity against the A-549 cell line, with an IC(50) value of 16.77 µM.

Cyclovirobuxine D, a cardiovascular drug from traditional Chinese medicine, alleviates inflammatory and neuropathic pain mainly via inhibition of voltage-gated Cav3.2 channels.

Cyclovirobuxine D (CVB-D), the main active constituent of traditional Chinese medicine Buxus microphylla, was developed as a safe and effective cardiovascular drug in China. B. microphylla has also been used to relieve various pain symptoms for centuries. In this study, we examined and uncovered strong and persistent analgesic effects of cyclovirobuxine D against several mouse models of pain, including carrageenan- and CFA-induced inflammatory pain and paclitaxel-mediated neuropathic hypersensitivity. Cyclovirobuxine D shows comparable analgesic effects by intraplantar or intraperitoneal administration. Cyclovirobuxine D potently inhibits voltage-gated Cav2.2 and Cav3.2 channels but has negligible effects on a diverse group of nociceptive ion channels distributed in primary afferent neurons, including Nav1.7, Nav1.8, TRPV1, TPRA1, TRPM8, ASIC3, P2X2 and P2X4. Moreover, inhibition of Cav3.2, rather than Cav2.2, plays a dominant role in attenuating the excitability of isolated dorsal root ganglion neurons and pain relieving effects of cyclovirobuxine D. Our work reveals that a currently in-use cardiovascular drug has strong analgesic effects mainly via blockade of Cav3.2 and provides a compelling rationale and foundation for conducting clinical studies to repurpose cyclovirobuxine D in pain management.

Pyranone Derivatives With Antitumor Activities, From the Endophytic Fungus Phoma sp. YN02-P-3.

Two new pyranone derivatives phomapyrone A (2) and phomapyrone B (3), one new coumarin 11S, 13R-(+)-phomacumarin A (1), three known pyranones (4-6), together with three known amide alkaloids fuscoatramides A-C (7-9), as well as 9S, 11R-(+)-ascosalitoxin (10) were isolated from the endophytic fungus Phoma sp. YN02-P-3, which was isolated from the healthy leaf tissue of a Paulownia tree in Yunnan Province, China. Their structures were elucidated using extensive NMR spectroscopic and HRESIMS data and by comparing the information with literature data. In addition, all compounds were tested for their cytotoxicity activity against human tumor cell lines, and the results showed that new compounds 1-3 showed moderate inhibitory activity against the HL-60 cell line with IC50 values of 31.02, 34.62, and 27.90 µM, respectively.

Caged polycyclic polyprenylated acylphloroglucinols as Cav3.2 low voltage-gated Ca2+ channel inhibitors from Hypericum curvisepalum.

Five caged polycyclic polyprenylated acylphloroglucinols including an unprecedented octahydro-2,5-methanoindene skeleton (1) were discovered from Hypericum curvisepalum. Biologically, 1 and 2 are potent Cav3.2 T-type Ca2+ channel inhibitors with negligible effect on the cardiovascular antitarget, the human ether-à-go-go-related gene potassium channel. Additionally, 2 indicates strong antinociception in the mouse acetic acid writhing test.

Cycloartane triterpenoids from Cimicifuga yunnanensis induce apoptosis of breast cancer cells (MCF7) via p53-dependent mitochondrial signaling pathway.

The present study was carried out to investigate the antitumor activity of five cycloartane triterpenoids isolated from Cimicifuga yunnanensis on the breast cancer cell line MCF7 and its corresponding drug resistant subline R-MCF7, including cimigenol-3-O-ß-D-xylopyranoside (compound 1), 25-O-acetylcimigenol-3-O-ß-D-xylopyranoside (compound 2), 25-chlorodeoxycimigenol-3-O-ß-D-xylopyranoside (compound 3), 25-O-acetylcimigenol-3-O-α-L-arabinopyranoside (compound 4) and 23-O-acetylcimigenol-3-O-ß-D-xylopyranoside (compound 5). The results showed that compounds 2-5 have relatively high antitumor activity on both MCF7 and R-MCF7 cells. The involvement of apoptosis as a major cause of cycloartane triterpenoids-induced cell death was further confirmed. The results of RT-PCR showed that compounds 2-5 increased the expression of p53 and bax, which led to the loss of mitochondrial potential and then resulted in the activation of caspase-7. These findings collectively demonstrated that compounds 2-5 induced apoptosis of MCF7 via p53-dependent mitochondrial pathway.

Diterpenoid Alkaloids from the Aerial Parts of Aconitum flavum Hand.-Mazz.

Sixteen diterpenoid alkaloids (DAs), including six aconitine-type alkaloids (5 and 9 - 13), seven 7,17-seco-aconitine-type alkaloids (1 - 4, 6 - 8), two napelline-type alkaloids (14 and 15) as well as one veatchine-type alkaloid (16), were isolated from the aerial parts of Aconitum flavum Hand.-Mazz. In which, flavumolines A - D (1 - 4) were four new ones, and flavumoline E (5) was reported as natural compound for the first time. Their chemical structures were elucidated by the analysis of extensive spectroscopic data. The inhibitory activities of these isolates on Cav3.1 low voltage-gated Ca2+ channel, NO production in LPS-activated RAW264.7cells, five human tumor cell lines, as well as acetylcholinesterase (AChE) were tested.

Isoprenylated Flavonoids as Cav3.1 Low Voltage-Gated Ca2+ Channel Inhibitors from Salvia digitaloides.

Saldigones A-C (1, 3, 4), three new isoprenylated flavonoids with diverse flavanone, pterocarpan, and isoflavanone architectures, were characterized from the roots of Salvia digitaloides, together with a known isoprenylated flavanone (2). Notably, it's the first report of isoprenylated flavonoids from Salvia species. The structures of these isolates were elucidated by extensive spectroscopic analysis. All of the compounds were evaluated for their activities on Cav3.1 low voltage-gated Ca2+ channel (LVGCC), of which 2 strongly and dose-dependently inhibited Cav3.1 peak current.

New ent-kaurane diterpenes from the roasted arabica coffee beans and molecular docking to α-glucosidase.

Ten new (1-10) and five known (11-15) ent-kaurane diterpene derivatives were identified from the roasted beans of coffea arabica. Their structures were established by extensive spectroscopic analysis including 1D, 2D NMR (HSQC, HMBC, COSY, and ROESY), HRESIMS, and X-ray diffraction analysis. Compounds 1-3 were three types of rearranged ent-kaurane diterpenes, and compounds 4 and 5 were diterpene esters with a rare 6-hydroxyhexanoyl at C-17. Compounds 6, 8, 14, and 15 showed moderate inhibitory effect on α-glucosidase with IC50 values of 149.92 ± 2.52, 23.23 ± 1.03, 54.58 ± 4.21, 54.16 ± 3.95 µM, respectively, compared to the positive control (60.71 ± 16.45 µM). The results of activity assay showed that diterpenes with the double bond between C-15 and C-16 exhibited stronger α-glucosidase inhibitory activity. Further molecular docking experiments were adopted to discuss the mechanism of activity.

Discovery of novel coffee diterpenoids with inhibitions on Cav3.1 low voltage-gated Ca2+ channel.

Seven new (1-4, 6-8) diterpenoids with rare skeletons and seven known ones (9, 12, 17, 18 and 23-25) were isolated from roasted beans of Coffea arabica L. Together with previously obtained diterpenoids, a total of 26 molecules (1-25, 4a) were evaluated their activities on Cav3.1 low voltage-gated Ca2+ channel. Compounds 1, 3, 6, 7, 12, 13, 17, 19 and 24 exhibited noticeable Cav3.1 inhibitions (41.2%-96.1%) at 10 µM. The IC50 values of 1, 6, 7, 12, 13, 17 and 24 are 2.9, 2.3, 0.68, 14.8, 11.6, 6.1 and 6.8 µM, respectively. The ring moiety at C-18 and C-19, and esterification of OH-17 with long-chain fatty acids seem important for their activities. Further studies indicated that 1 and cafestol may act on different binding sits with the Cav3.1 blocker Z944, which is in clinical trial. Significantly, the present study initially shows that coffee diterpenoids are potential natural resources for Cav3.1 inhibitors.